molecules Review Characteristics of the Proteolytic Enzymes Produced by Lactic Acid Bacteria Marek Kieliszek * , Katarzyna Pobiega , Kamil Piwowarek and Anna M. Kot Department of Food Biotechnology and Microbiology, Institute of Food Sciences, Warsaw University of Life Sciences—SGGW, Nowoursynowska 159 C, 02-776 Warsaw, Poland; [email protected] (K.P.); [email protected] (K.P.); [email protected] (A.M.K.) * Correspondence: [email protected] or [email protected]; Tel.: +48-22-593-76-57; Fax: +48-22-593-76-81 Abstract: Over the past several decades, we have observed a very rapid development in the biotech- nological use of lactic acid bacteria (LAB) in various branches of the food industry. All such areas of activity of these bacteria are very important and promise enormous economic and industrial successes. LAB are a numerous group of microorganisms that have the ability to ferment sugars into lactic acid and to produce proteolytic enzymes. LAB proteolytic enzymes play an important role in supplying cells with the nitrogen compounds necessary for their growth. Their nutritional requirements in this regard are very high. Lactic acid bacteria require many free amino acids to grow. The available amount of such compounds in the natural environment is usually small, hence the main function of these enzymes is the hydrolysis of proteins to components absorbed by bacterial cells. Enzymes are synthesized inside bacterial cells and are mostly secreted outside the cell. This type of proteinase remains linked to the cell wall structure by covalent bonds. Thanks to advances in enzymology, it is possible to obtain and design new enzymes and their preparations that can be Citation: Kieliszek, M.; Pobiega, K.; widely used in various biotechnological processes. This article characterizes the proteolytic activity, Piwowarek, K.; Kot, A.M. describes LAB nitrogen metabolism and details the characteristics of the peptide transport system. Characteristics of the Proteolytic Potential applications of proteolytic enzymes in many industries are also presented, including the Enzymes Produced by Lactic Acid food industry. Bacteria. Molecules 2021, 26, 1858. https://doi.org/10.3390/molecules Keywords: proteolytic enzymes; lactic acid bacteria; proteolysis 26071858 Academic Editor: Stefan Janecek 1. Introduction Received: 3 March 2021 The development of global industry and the achievement of a high level of knowledge Accepted: 23 March 2021 Published: 25 March 2021 on the nature of enzymes have allowed enzymes to be used for the production of high- quality consumer goods, thus proving their industrial significance. Enzymes perform Publisher’s Note: MDPI stays neutral an extremely important function; that is, they act as biocatalysts, mediate all anabolic with regard to jurisdictional claims in and catabolic pathways, and reduce the activation energy of biochemical reactions [1]. published maps and institutional affil- Although the catalytic activity of enzymes has been known to humans for many centuries, iations. recent advances in the global biotechnology industry have enabled a wider use of these biocatalysts. Currently, hydrolytic enzymes are very popular and widely used [2,3]. The lactic acid bacteria group consists of species that are among the best-studied microorganisms. What is more, proteolysis is one of the particular physiological properties of lactic acid bacteria of which important knowledge has been attained. The proteolytic Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. system of lactic acid bacteria is related to the utilization of casein and supplies the cells with This article is an open access article essential amino acids during their growth in milk. Lactic acid bacteria and their proteolytic distributed under the terms and system play a crucial role when it comes to the organoleptic properties of fermented milk conditions of the Creative Commons products. Except for the processing of nutrients, cellular proteolysis is responsible for Attribution (CC BY) license (https:// controlling the quality of polypeptides and regulatory mechanisms by maintaining an creativecommons.org/licenses/by/ appropriate level of regulatory proteins, and, when necessary, removing them [4]. The 4.0/). proteolytic system of lactic acid bacteria comprises proteinases, peptidases, and specific Molecules 2021, 26, 1858. https://doi.org/10.3390/molecules26071858 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 1858 2 of 15 transport proteins. Proteinases cleave casein into peptides, then peptidases (intracellular) degrade peptides to amino acids and smaller peptides. Transport systems are responsible for transferring amino acids and peptides across the cytoplasmic membrane [5]. The increasing demand for proteolytic enzymes has prompted the need to seek new living organisms that can serve as sources of these enzymes, as well as to perform in- depth analysis of the already available genetic material. Presently, the vast majority of enzymes are produced by recombinant microbial strains, because of which biocatalysts are much cheaper than the proteins isolated from representatives of natural producers [6,7]. Many researchers have focused their interest on understanding the activity, regulation, and physiological function of the proteolytic system of lactic acid bacteria [8–11]. This manuscript depicts the review of the components of the proteolytic system in lactic acid bacteria that are associated with food fermentations. Special emphasis has been placed on, for example, the structure of extracellular proteinases, the peptide transport system, regulation of the proteolytic system, and industrial use of proteolytic enzymes, including by the dairy, baking and brewing industries. 2. Characteristics of Lactic Acid Bacteria Lactic acid bacteria (LAB) are a group of microbes with increasing importance in biotechnology. They are widely used in milk processing. Traditionally, the use of LAB was mainly limited to the dairy, fermentation, fruit and vegetable, and animal feed industries. The importance of lactic acid bacteria is constantly growing because they are considered safe for humans and animals (Generally Recognized As Safe—GRAS) and show many beneficial effects on human health [12–16]. LAB are a large group of bacteria with the ability to ferment sugars into lactic acid. Lactic acid fermentation is an intracellular, anaerobic enzymatic process of converting sugars to lactic acid, which is one of the approaches to obtain the energy necessary for cell life processes in anaerobic conditions [17–19]. Morphologically, lactobacilli are not a homogeneous group. They include rods arranged in chains, such as the genus Lacticas- eibacillus, Carnobacterium, Weissella; cocci arranged in chains, such as species of Lactococcus, Enterococcus, Vagococcus; and Oenococcus (known as Leuconostoc oeni), which was formerly referred to as Leuconostoc, and are arranged in the form of tetrads, such as the genus Tetragenococcus. These are relatively anaerobic, gram-positive, and nonspore-producing bacteria that usually do not have flagellas [12,13,17,20,21]. LAB can produce proteolytic enzymes inside the cell as well as in the external en- vironment. The optimum pH for the development and growth of proteolytic bacteria in milk is 7–7.5. A low temperature inhibits the growth and development of these bacteria and decreases their enzymatic activity. Temperatures above that are optimal for bacterial growth rapidly inactivate proteolytic enzymes. In some cases, irreversible loss of prote- olytic enzyme activity is observed after prolonged storage at low temperatures [22–24]. Many proteolytic bacteria have psychrotrophic properties. It should be noted that these bacteria can degrade milk proteins. This aspect has been observed in both bacilli and lactic streptococci, although to a lesser extent than that in typical proteolytic bacteria such as Proteus, Pseudomonas, Alcaligenes, Acinetobacter, Bacillus, Clostridium, and some strains of Micrococcus [25]. LAB can degrade proteins, as exemplified by casein. The final products of this decom- position are most often the amino acids used by LAB as a source of nitrogen. Thermophilic lactobacilli species have stronger proteolytic abilities than rods or streptococci; however, each species has strains with highly varying activity in this respect. Depending on the species, subspecies, and even the strain, LAB show very diverse proteolytic activity. Among the cocci, the most active species are Lactococcus salivarius subsp. thermophilus (formerly named Streptococcus salivarius subsp. thermophilus) and Lc. lactis subsp. cremonis, while the least active is Lc. lactis subsp. lactis. Of the lactobacilli used in the dairy industry, the most proteolytically active species are Lacticaseibacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, Lb. helveticus, and Lb. acidophilus, while the least active species is Molecules 2021, 26, 1858 3 of 15 Lactiplantibacillus plantarum. In general, the activity of rods is greater than that of lactic streptococci [24,26–29]. When discussing the proteolytic enzymes of LAB, it is worth mentioning lactococcal strains. Proteolysis of casein forms so-called bitter peptides, which are the cause of the bitter taste of dairy products [30]. 3. Enzymatic Activities of Proteases Proteolytic enzymes can be classified as exopeptidases or endopeptidases (proteinases) (Figure1). The first group cleave the peptide bond proximal to the amino or carboxy termini of the substrate.